Gene encoding resistance to acetolactate synthase-inhibiting herbicides

ABSTRACT

A mutant acetolactate synthase (ALS) enzyme that confers cross-resistance to all sulfonylurea, imidazolinone, pyrimidinyloxybenzoate, triazolopyrimidine and sulfonylamino-carbonyl-triazolinone herbicides is provided. The mutant enzyme contains an aspartic acid to glutamic acid substitution mutation at a newly identified conserved region of the ALS enzyme. A gene encoding the enzyme is also provided, as are transgenic plants that have been genetically engineered to contain and express the gene. The transgenic plants are cross-resistant to sulfonylurea, imidazolinone, pyrimidinyloxybenzoate, triazolopyrimidine and sulfonylamino-carbonyl-triazolinone herbicides.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to herbicide resistance. In particular,the invention provides a mutant acetolactate synthase (ALS) gene thatconfers cross-resistance to all sulfonylurea, imidazolinone,pyrimidinyloxybenzoate, triazolopyrimidine, andsulfonylamino-carbonyl-triazolinone herbicides.

2. Background of the Invention

Herbicides have simplified weed management in agriculture and provide ahighly effective means of keeping weed populations at acceptable levels.However, crop sensitivity to numerous herbicides limits the use of theseherbicides to tolerant crops only. Certain herbicides currentlyregistered for use in crops still result in injury even at normal userates. Crop injury increases when higher application rates are requiredto manage large weeds or heavy infestations that are beyond control withnormal use rates. In extreme situations, the only effective herbicidesavailable may result in significant crop injury. Furthermore, residualherbicides remaining in the soil are often a problem with rotation to asensitive crop the following season, which may hinder the use ofeffective herbicides based on rotational restrictions. Modification ofcrop plants to create herbicide resistance has been an effective tool toincrease weed control, minimize crop injury, allow applications ofherbicides in crops with previous sensitivity, reduce herbicide inputs,and make use of more environmentally sound herbicide options. Transgeniccrops resistant to a specific herbicide have been developed bytransformation with target enzymes that are insensitive to a specificherbicide.

Acetolactate synthase (ALS) is an enzyme that catalyzes the initial stepin the branched chain amino acid biosynthetic pathway. ALS is the targetsite of several classes of unrelated herbicide chemistries, includingsulfonylureas (SU), imidazolinones (IMI), pyrimidynyloxybenzoates (POB),triazolopyrimidines (TP), and sulfonylamino-carbonyl-triazolinones(Table 1). Currently, ALS-inhibiting herbicides comprise the largestmode-of-action group in use due to broad-spectrum weed control in avariety of crops at very low application rates. In addition,ALS-inhibiting herbicides have very low mammalian toxicity. Thesecharacteristics have increased the importance of these herbicides inproduction agriculture and have attracted the development ofALS-resistant crops.

A single nucleotide mutation in the ALS enzyme is capable of conferringherbicide specific resistance. Mutations have been identified in fivehighly conserved domains along the DNA sequence coding for the ALSenzyme in higher plants. Each domain contains a single variable residue,that when substituted, confers resistance to specific ALS-inhibitingherbicides. In most cases, a single substitution results in target-sitecross-resistance differences between ALS-inhibiting herbicidechemistries (Table 2). A substitution reported at Ala₁₃₃ in domain C ofcommon cocklebur resulted in resistance to IMI herbicides only. Theidentical mutation was found in a commercial field corn hybrid, ICI 8532IT, and sugar beet line Sur, which are crops resistant to only IMIherbicides (Bernasconi et al., J. Biol. Chem. (1995) 270:17381-17385;Wright et al., Weed Sci. (1998) 46:13-23). Substitutions at Pro₁₉₇ indomain A have resulted in a high level of resistance to SU herbicideswith little or no resistance to IMI herbicides (Guttieri et al., WeedSci. (1992) 40:670-676; Guttieri et al., Weed Sci (1995) 43:175-178;Boutsalis et al., Pestic. Sci. (1999) 55:507-516). A domain E mutationof Ser₆₇₀ to Asp resulted in a high level of resistance to IMIherbicides with low SU resistance (Devine and Eberlein, HerbicideActivity: Toxicology, Biochemistry and Molecular Biology (1997)159-185).

High-level cross-resistance between ALS-herbicide chemistries has beenshown previously with field isolated common cocklebur (Xanthiumstrumarium) biotypes exposed to several years of ALS selection pressure(Bemasconi et al., J. Biol. Chem. (1995) 270:17381-17). The isolatedprotein from one resistant biotype had a Trp₅₅₂ to Leu mutation ascompared to the susceptible population. This mutation corresponded tothe Trp₅₄₂ to Leu mutation in a commercial corn hybrid, Pioneer 3180 IR,which exhibited broad-range tolerance to ALS-inhibiting herbicides. Asecond common cocklebur field isolate had a substitution of Ala₁₈₃ toVal in Domain D that conferred similar cross-resistance patterns to themutation found in domain B (Woodworth et al., Plant Physiol. (1996)111:415). TABLE 1 Representative examples of sulfonylurea,imidazolinone, pyrimidinyloxybenzoate, and triazolopyrimidineALS-inhibiting herbicides and corresponding chemical names.ALS-Inhibitor Family Common Name Chemical Name Sulfonylurea chlorimuron2-[[[[(4-chloro-6- methoxy-2-pyrimidinyl)- amino]carbonyl]amino]-sulfonyl]benzoic acid thifensulfuron 3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2- yl)amino]carbonyl]- amino]sulfonyl]-2-thiophenecarboxylic acid trifloxysulfuron N-[(4,6-dimehoxy-2-pyrimidinyl)carbamoyl]- 3-(2,2,2-trifluoroethoxy)- pyridin-2-sulfonamidenicosulfuron 2-[[[[(4,6- dimethoxy-2-pyrimidinyl)- amino]carbonyl]-amino]sulfonyl]-N,N- dimethyl-3-pyridine- carboxamide Imidazolinoneimazethapyr 2-[4,5-dihydro-4-methyl- 4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl- 3-pyridinecarboxylic acid imazaquin2-[4,5-dihydro-4-methyl- 4-(1-methylethyl)-5-oxo- 1H-imidazol-2-yl]-3-quinolinecarboxylic acid imazapyr (±)-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)- 5-oxo-1H-imidazol-2-yl]-3-pyridinecarboxylic acid Pyrimidinyloxy- pyrithiobac2-chloro-6-[(4,6-dimethoxy- benzoate 2-pyrimidinyl)thio]benzoic acidTriazol- cloransulam 3-chloro-2-[[(5-ethoxy- pyrimidine7-fluoro[1,2,4]triazolo[- 1,5-c]pyrimidin-2yl)- sulfonyl]amino]benzoicacid flumetsulam N-(2,6-difluorophenyl)-5- methyl[1,2,4]triazolo-[1,5-a]pyrimidine-2- sulfonamide Sulfonylamino- flucarbazone4,5-dihydro-3-methoxy-4- carbonyl- methyl-5-oxo-N-[[2- triazolinones(trifluoromethoxy)phenyl]- sulfonyl]-1H-1,2,4- triazole-1-carboxamidepropoxycarbazone methyl 2-[[[(4,5- dihydro-4-methyl-5-oxo-3-propoxy-1H-1,2,4-triazol- 1-yl)carbonyl]amino]- sulfonyl]benzoate

TABLE 2 Common ALS mutations and corresponding levels of resistanceconferred to SU, IMI, and TP herbicides (Devine and Shukla, Crop Prot.(2000) 19:881-889). Resistance Level Mutation Domain Domain Sequence SUIMI TP Reference Ala₁₂₂ to Thr C VFAYPGGASMEIHQALTRS Low High LowBernasconi et al. (1995) (SEQ ID NO: 8) zero zero Pro₁₉₇ to Ala AAITGQVPRRMIGT High Zero Mod Boutsalis et al. (1999) (SEQ ID NO: 9) lowPro₁₉₇ to Thr High Low — Guttieri et al. (1995) zero Pro₁₉₇ to His HighMod Low Guttieri et al. (1992) Pro₁₉₇ to Leu High Mod High Guttieri etal. (1995) low Pro₁₉₇ to Arg High — — Guttieri et al. (1995) Pro₁₉₇ toIle High Mod Mod Boutsalis et al. (1999) low low Pro₁₉₇ to Gln High — —Guttieri et al. (1995) Pro₁₉₇ to Ser High Zero High Guttieri et al.(1995) Ala₂₀₅ to Asp D AFQETP High — — Hartnett et al. (1990) (SEQ IDNO: 10) Woodworth et al. (1996)(2) Trp₅₉₁ to Leu B QWED High High HighBoutsalis et al. (1999) (SEQ ID NO: 11) Ser₆₇₀ to Asp E IPSGG Low HighZero Devine and Eberlein (1997) (SEQ ID NO: 12)

ALS-resistant crops currently marketed provide herbicide resistance toonly a single class of ALS-inhibiting herbicides, either imidazolinoneor sulfonylurea classes. There is an ongoing need to develop herbicideresistant crops, and it would be particularly desirable to develop cropswith resistance to more than one herbicide. The prior art has thus farfailed to meet this need.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a functional, mutant ALSenzyme that is broadly resistant to ALS-inhibiting herbicidechemistries. Transgenic plants that have been genetically engineered tocontain and express a gene encoding the enzyme are thus able to grow andreproduce even after the application of two or more herbicides (eventhose representing different herbicide families) to which the mutant ALSconfers resistance. In contrast, other plants (e.g. weeds) that may beresistant to one family of the herbicides, but are not resistant toother families of ALS-inhibiting herbicides will be inhibited in theirgrowth and reproduction after the application of two or more herbicides.In the mutant enzyme, ALS resistance is conferred by a single amino acidmutation in a conserved region previously unreported along the ALS genein higher plants. The ALS enzyme of the present invention iscross-resistant to at least four classes of structurally unrelatedALS-inhibiting herbicide chemistries, including imidazolinones,sulfonylureas, pyrimidinyloxybenzoates, triazolopyrimidines, andsulfonylamino-carbonyl-triazolinones. Together, these classes comprisethe largest mode-of-action herbicide group, representing over 50commercial herbicides used in all major crops (eg. corn, wheat, soybean,rice, cotton, and canola) and a wide range of minor crops. This mutationcreates an ALS enzyme with resistance to any ALS enzyme-inhibitingherbicide and offers the possibility of creating herbicide-resistantcrops with cross-resistance to all herbicides in these groups.

The present invention thus provides a substantially purifiedacetolactate synthase (ALS) enzyme that confers, in a plant,cross-resistance to multiple herbicides. In one embodiment of theinvention, the sequence of the ALS enzyme is SEQ ID NO: 1, or a fragmentthereof with ALS activity. At least two of the multiple herbicides maybe sulfonylurea, imidazolinone, pyrimidinyloxybenzoate,triazolopyrimidine or sulfonylamino-carbonyl-triazolinone herbicides.

The invention also provides a substantially purified ALS gene encodingan ALS enzyme that confers, in a plant, cross-resistance to multipleherbicides. In one embodiment, the gene is SEQ ID NO: 2 or a fragmentthereof encoding a polypeptide with ALS activity. At least two of themultiple herbicides may be sulfonylurea, imidazolinone,pyrimidinyloxybenzoate, triazolopyrimidine orsulfonylamino-carbonyl-triazolinone herbicides.

The invention also provides a method of conferring cross-resistance tomultiple herbicides to a plant. The method includes the step ofintroducing into the plant an expressible gene encoding an ALS enzymethat exhibits cross-resistance to multiple herbicides. The step ofintroducing the gene into the plant confers cross-resistance to multipleherbicides to the plant. The gene may be SEQ ID NO: 1, or a fragmentthereof that encodes a polypeptide having ALS activity.

The invention also provides a transgenic plant that is cross-resistantto multiple herbicides. The transgenic plant is comprised of a hostplant that contains an expressible gene that is not naturally present inthe plant, and the gene encodes an ALS enzyme that conferscross-resistance to multiple herbicides. The gene may be SEQ ID NO:2, ora fragment thereof that encodes a polypeptide having ALS activity. Themultiple herbicides may be sulfonylurea, imidazolinone,pyrimidinyloxybenzoate, triazolopyrimidine orsulfonylamino-carbonyl-triazolinone herbicides. The transgenic plant maybe, for example, Arabidopsis, corn, cotton, soybean, rice, wheat, or aforage crop. The ALS enzyme may have an aspartic acid to glutamic acidsubstitution at position six of a conserved sequence GVRFDDRVTGK (SEQ IDNO: 6).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and B. R11-AMACH sequences. A) nucleotide sequence; B) primaryamino acid sequence.

FIG. 2A and B. S-AMACH sequences. A) nucleotide sequence; B) primaryamino acid sequence.

FIG. 3. Amino acid sequence alignment of R11-AMACH and S-AMACH ALS gene.The mutation (D to E) is indicated on top of the alignment (#) atposition 375 within the highlighted region.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides a mutant acetolactate (ALS) enzyme thatconfers, when produced by a plant, cross-resistance to multipleherbicides. The enzyme contains a single amino acid substitution (aspartic acid to glutamic acid, in a newly identified conserved regionof the ALS enzyme. The conserved region has the primary amino acidsequence GVRFDDRVTGK, (SEQ ID NO: 6) and the D to E substitution occursat aspartic acid at position 6, resulting in a mutant conserved sequenceGVRFDERVTGK, (SEQ ID NO: 7). In smooth pigweed (Amaranthus hybridus L.)ALS, the D to E substitution is at residue 375; in Arabidopsis, the D toE substitution is at residue 376. Those of skill in the art willrecognize that the precise position of the substitution in a full lengthALS enzyme may vary from species to species, or from variety to varietydue to genetic variation.

However, the mutation is located at amino acid position 6 of theconserved sequence GVRFDDRVTGK, (SEQ ID NO: 6). The “conserved sequence”itself may vary slightly depending on the source, and in particular mayhave conservative amino acid substitutions, but will generally be in therange of about 90 to 100% homologous to (SEQ ID NO: 6).

Surprisingly, the mutant ALS enzyme, when produced in a plant, rendersthe plant cross-resistant to multiple classes of herbicides, includingimidazolinones, sulfonylureas, pyrimidinyloxybenzoates,triazolopyrimidines and sulfonylamino-carbonyl-triazolinones. Theinvention also provides a mutant gene that encodes the cross-resistantALS enzyme, as well as transgenic plants that have been geneticallyengineered to contain the mutant gene and which thus produce afunctional mutant enzyme, and a method for transforming plants with themutant gene. Such transgenic plants display cross-resistance to multipleherbicides and are able to grow and reproduce even after the applicationof two or more of the herbicides to which they are cross-resistant.

In a preferred embodiment of the invention, the nucleotide sequence thatencodes the cross-resistant ALS enzyme is that which is shown in FIG. 1A(SEQ ID NO: 1). The corresponding amino acid sequence is given in FIG.1B (SEQ ID NO: 2). However, those of skill in the art will recognizethat various permutations of the nucleic acid sequence of SEQ ID NO: 1may also be used to encode an enzyme of the present invention. Theseinclude but are not limited to: shorter portions of the DNA moleculewhich encode truncated ALS enzymes that still possess ALS activity andexhibit cross-resistance to multiple herbicides; nucleic acid sequencesthat contain various substitutions that, due to the redundancy of thegenetic code, still encode an enzyme identical to SEQ ID NO: 2; nucleicacid sequences that are substantially as in SEQ ID NO: 2 but which havebeen altered for any reason, such as to introduce a convenientrestriction enzyme cleavage site, to alter the tertiary structure of theDNA molecule, etc.; various nucleic acid sequences that aresubstantially homologous to SEQ ID NO: 1 (e.g. that display from about70% to about 100% homology, and preferably about 80% to 100% homology,and most preferably about 90% to 100% homology) to SEQ ID NO: 1, butstill encoding an enzyme with ALS activity and which displays multipleherbicide cross-resistance as described herein. All such DNA molecules,as well as any vectors which include the DNA molecules, are intended tobe encompassed within the scope of the present invention.

The invention further encompasses RNA molecules which encode a mutantALS enzyme of the present invention, for example, mRNA moleculestranscribed from a gene encoding an ALS enzyme of the present invention.

Accordingly, the invention also contemplates mutant ALS enzymes thatdisplay cross-resistance to multiple herbicides and which have a primaryamino acid sequence as in SEQ ID NO: 2. Alternatively, various relatedbut non-identical polypeptide sequences are also contemplated by thepresent invention, e.g. polypeptides which possess about 70 to 100%homology, or preferably 80 to 100% homology, or most preferably 90 to100% homology to SEQ ID NO: 2, so long as the related polypeptidedisplays ALS activity, and exhibits cross-resistance to multipleherbicides. Changes to the sequence may be made for any reason, and mayinvolve conservative or nonconservative amino acid substitutions, oramino acid additions or deletions. For example, residues may be changedby well-known genetic engineering techniques in order to introduce oreliminate sequences susceptible to cleavage by proteases, tonon-enzymatic deamidation reactions, to various post-translationalmodification reactions (e.g. glycosylation, acetylation, etc.), toenhance solubility of the polypeptide, etc. Such mutant ALS enzymes maybe approximately a fall length polypeptide such as that of SEQ ID NO: 2.Alternatively, the mutant ALS enzyme may be a truncated version orfragment of the polypeptide which retains ALS activity and multipleherbicide resistance (e.g. a shorter polypeptide that is based on theprimary sequence of the fall length ALS gene but has, for example, aportion of the carboxy or amino terminal residues deleted, or which hasa portion of intervening sequences between the carboxy and amino terminideleted. Such a fragment would in general possess about 70 to 100%homology to the region of full length ALS to which it corresponds (i.e.the primary sequence of ALS that was not deleted), or preferably about80 to 100% homology, and most preferably about 90 to 100% homology.Further, the sequence of the mutant ALS enzyme may be geneticallyengineered to contain other useful sequences, e.g. sequences which serveto target the polypeptide to a location within the cell or within theplant, sequences which facilitate isolation of the protein (e.g. anamino acid tag), and the like. In addition, chimeric fusion proteins inwhich the mutant ALS enzyme is translated in tandem with or otherwisejoined to a related or non-related protein, examples of which includebut are not limited to proteins or polypeptides which facilitatetracking of the ALS enzyme (e.g. green fluorescent protein, etc.) orproteins which confer some other useful property to the enzyme or plant,such as antibiotic resistance, or markers such as β-glucoronidaase(GUS), luciferase, β-galactosidase, chloramphenicol acetyl transferase(CAT), octopine, nopaline synthase, NPT-II, etc. All such variations ofthe mutant ALS enzyme depicted in SEQ ID NO: 2 are intended to beencompassed by the present invention, so long as the variant enzymesdisplay ALS activity and cross-resistance to multiple herbicides.

By “displays ALS activity” we mean that the mutant, geneticallyengineered enzyme exhibits at least about 50% or more of the level ofactivity of the corresponding wild type enzyme, when tested understandard conditions for testing ALS activity. Typically, the measurementof ALS activity utilizes a discontinuous colorimetric assay as describedby Singh et al., (1988). The assay involves combining enzyme, pyruvate,cofactors, and other additives, followed by a fixed time incubation. Thereaction is terminated by addition of sulfuric acid and heated toconvert acetolactate to acetoin. The acetoin is converted to a coloredcomplex upon addition of creatine and α-naphthol, as described byWesterfield, (1945). The absorbance of the reaction mixture is measuredat 525 nm.

By “herbicide resistance” we mean an inherited ability of a plant tosurvive and reproduce following treatment with a dose of herbicide thatwould be lethal to the wild type. This definition includes plantsrendered resistant through genetic engineering. By “cross-resistance” or“cross-resistance to multiple herbicides” we mean herbicide resistanceto two or more herbicides that have the same general mechanism ofaction, for example, the inhibition of an enzyme such as ALS. This is incontrast to “multiple herbicide resistance” which is understood to meanherbicide resistance to two or more herbicides that have completelydifferent mechanisms of action.

Herbicides of interest for the present application include but are notlimited to sulfonylureas, imidazolinones, pyrimidinyloxybenzoates,triazolopyrimidines, and sulfonylamino-carbonyl-triazolinones, as wellas any other classes of herbicides that act through inhibition of theALS enzyme.

The ALS mutant enzyme of the present invention and the nucleic acidencoding the ALS mutant enzyme of the present invention may be providedin a substantially purified form. By “substantially purified” we meanthat the molecule is substantially free of other contaminating matter(such as molecules of other protein, nucleic acids, plant tissue, etc.)which might be associated with the enzyme or nucleic acid of the presentinvention prior to purification. Those of skill in the art willrecognize the standards typically used for assessing purification of anenzyme or nucleic acid, and the means for carrying out such anassessment (e.g. analysis via chromatography, gels, mass spectroscopy,nuclear magnetic resonance, measurement of activity, etc.). The level ofpurification will generally be greater than about 60%, and preferablygreater than about 75%, and most preferably from about 90 to 100% pure,based on, for example, a weight to weight basis of enzyme or nucleicacid to enzyme or nucleic acid plus contaminant(s). The presentinvention also provides transgenic plants that have been geneticallyaltered (i.e. genetically engineered) to contain and express a geneencoding a mutant ALS enzyme that confers to the plants cross-resistanceto multiple herbicides. The expressible gene is not naturally present inthe plant, and it is typically introduced into the plant by any of manywell-known genetic engineering techniques. The invention furtherprovides a method of conferring herbicide cross-resistance to a plant byintroducing into the plant a gene encoding a mutant ALS enzyme of thepresent invention. The methodology for creating transgenic plants iswell developed and well known to those of skill in the art. For example,dicotyledon plants such as soybean, squash, tobacco (Lin et al. 1995),and tomatoes can be transformed by Agrobacterium-mediated bacterialconjugation. (Miesfeld, 1999, and references therein). In this method,special laboratory strains of the soil bacterium Agrobacterium are usedas a means to transfer DNA material directly from a recombinantbacterial plasmid into the host cell. DNA transferred by this method isstably integrated into the genome of the recipient plant cells, andplant regeneration in the presence of a selective marker (e.g.antibiotic resistance) produces transgenic plants.

Alternatively, for monocotyledon plants, such as rice (Lin andAssad-Garcia, 1996), corn, and wheat which may not be susceptible toAgrobacterium-mediated bacterial conjugation, DNA may be inserted bysuch techniques as microinjection, electroporation or chemicaltransformation of plant cell protoplasts (Paredes-Lopez, 1999 andreferences therein), or particle bombardment using biolistic devices(Miesfeld, 1999; Paredes-Lopez, 1999; and references therein).Monocotyledon crop plants have now been increasingly transformed withAgrobacterium (Hiei, 1997) as well.

Further, development of the transgenic plant of the present inventionmay be carried out by the technique of homologous recombination, such asis described, for example, by Zhu et al., (2000).

In order to insert a gene encoding the mutant ALS enzyme of the presentinvention (i.e. into a host plant, the gene may be incorporated into asuitable construct such as a vector. Such vectors are well known tothose of skill in the art, and are used primarily to facilitate handlingand manipulation of the gene or gene fragment. Techniques formanipulating DNA sequences (e.g. restriction digests, ligationreactions, and the like) are well known and readily available to thoseof skill in the art. For example, see Brown, 1998 and Sambrook, 1989.Suitable vectors for use in the methods of the present invention arewell known to those of skill in the art. Such vectors include but arenot limited to pBC, pGEM, pUC, etc.

Further, such vector constructs may include various useful elements thatare necessary or useful for the expression of the gene. Examples of suchelements include promoters operably linked to the gene of interest (e.gstrong or inducible promoters), enhancer elements, genes for selectionsuch as antibiotic or other herbicide resistance genes (both cross- andmultiple-resistance genes), genes which encode factors necessary oruseful for effecting the transformation of plants with the gene ofinterest, terminators, targeting sequences, codes for affinity tags orantibody epitopes, etc. All such variations of the vector of the presentinvention are intended to be encompassed by the present invention, solong as the vector houses an ALS gene that encodes an ALS enzyme thatdisplays cross-resistance to multiple herbicides.

There are many host plants which could benefit by being transformed bythe methods of the present invention to exhibit resistance toherbicides. Such plants include both mono- and dicotyledon species.While the practice of the present invention is applicable to all plantspecies, it is especially useful for crop plants such as corn, wheat,soybean, cotton, rice, sorghum, canola, and the like. Further, the exactlevel of expression of the mutant ALS enzyme may vary somewhat fromplant to plant, or among species or varieties of plants that aretransformed with a mutant ALS gene of the present invention. However, ingeneral, any plant so transformed is intended to be within the scope ofthe present invention.

By “transgenic plant” we mean any segment or portion of a plant, atleast some cells of which contain a mutant ALS gene of the presentinvention, and express or previously expressed or are capable ofexpressing (e.g. upon further development) a mutant ALS enzyme of thepresent invention. Examples include but are not limited to: single plantcells; the stalks, roots, leaves and flowers of a plant; fruit or seedsproduced by the plant; vegetative organs such as rhizomes, stolen,bulbs, tubers, and corms; etc. All such portions of, products of,precursors of, etc. a transgenic plant are intended to be encompassed bythe present invention. Further, the term “plant” encompasses crop plantssuch as vegetables, grasses, bushes and trees that produce berries andfruits, ornamental plants (e.g. roses and other flowering plants), andforage crops (including alfalfa, clover, vetches, pasture and hay),grasses, grains, fiber crops, pulp trees, timber, etc.

The invention is further illustrated in the foregoing non-limitingexamples.

EXAMPLES Example 1 Characterization of ALS Resistance

Seeds from a smooth pigweed (Aniaranthus hybridus L.) population(R11-AMACH) were collected from a field in southeastern Pennsylvaniawhere extreme ALS-inhibitor herbicide selection pressure was imposedover a several year period within continuous soybean production.R11-AMACH was selected naturally with ALS-inhibiting herbicidesrepresentative of the SU, IMI, and TP herbicide chemistries.

To establish levels and patterns of ALS resistance, R11-AMACH and an ALSsusceptible smooth pigweed biotype (S-AMACH) were screened in thegreenhouse with various rates of the ALS-inhibiting herbicides,chlorimuron (SU), thifensulfuron (SU), imazethapyr (IMI), pyrithiobac(POB), and cloransulam-methyl (TP). Rates evaluated were based on alog10 scale that included 0, 1/100×, 1/10×, 1×, 10×, and 100×, where 1×corresponds to the normal use rate in the field. R11-AMACH respondeddifferently to the rate increase as compared to S-AMACH. With allherbicides applied, R11-AMACH showed high-levels of resistance based onthe response of the S-AMACH. Visual control, height, biomass, andbiomass reduction are presented separately for chlorimuron (Table 3),thifensulfuron (Table 4), imazethapyr (Table 5), pyrithiobac (Table 6),and cloransulam (Table 7). Evaluations and measurements were recorded 3weeks after herbicide treatment (WAT). Visual control was based on ascale of 0-99%, where 0% represents no control and 99% representscomplete control. Biomass represents plant dry weights recorded severaldays after plants were harvested. Biomass reduction was calculated basedon the amount of biomass reduced by herbicide treatment compared to theuntreated plant biomass.

Results show R11-AMACH resistance levels above 100 times the normal userate to both SU herbicides, chlorimuron and thifensulfuron, and to theTP herbicide, cloransulam-methyl. Resistance levels to IMI and POBherbicides, imazethapyr and pyrithiobac, respectively, were greater than10 times the normal use rate. Results indicated R11-AMACH hastarget-site cross-resistance to four classes of structurally unrelatedchemistries of ALS-inhibiting herbicides, namely SU, IMI, POB, and TP.TABLE 3 R11-AMACH and S-AMACH visual control, height, biomass, andbiomass reduction 3 WAT with various rates of chlorimuron (SU). VisualControl Height Biomass Biomass Reduction R11-AMACH S-AMACH R11-AMACHS-AMACH R11-AMACH S-AMACH R11-AMACH S-AMACH RATE % cm g % 0 0 0 33.830.5 3.80 3.43 0.0 0.0 1/100× 5 9 23.5 27.8 3.42 2.37 10.0 30.9 1/10× 620 26.5 14.3 3.66 1.65 3.7 52.0 1× 15 81 21.8 4.8 3.67 0.14 3.5 95.8 10×18 99 23.5 1.0 3.15 0.05 17.1 98.6 100× 39 99 14.8 1.3 1.64 0.11 56.896.7

TABLE 4 R11-AMACH and S-AMACH visual control, height, biomass, andbiomass reduction 3 WAT with various rates of thifensulfuron (SU).Visual Control Height Biomass Biomass Reduction R11-AMACH S-AMACHR11-AMACH S-AMACH R11-AMACH S-AMACH R11-AMACH S-AMACH RATE % cm g % 0 00 33.8 30.5 3.80 3.43 0.0 0.0 1/100× 4 22 36.0 25.0 4.21 1.76 −10.8 48.71/10× 9 47 32.5 11.0 4.45 0.85 −17.1 75.2 1× 22 98 23.0 2.0 3.48 0.078.4 98.0 10× 38 99 15.8 0.3 2.25 0.04 40.8 98.8 100× 64 99 6.8 0.5 0.580.03 84.7 99.1

TABLE 5 R11-AMACH and S-AMACH visual control, height, biomass, andbiomass reduction 3 WAT with various rates of imazethapyr (IMI). VisualControl Height Biomass Biomass Reduction R11-AMACH S-AMACH R11-AMACHS-AMACH R11-AMACH S-AMACH R11-AMACH S-AMACH RATE % cm g % 0 0 0 33.830.5 3.80 3.43 0.0 0.0 1/100× 2 12 32.8 25.3 4.11 1.98 −8.2 42.3 1/10× 958 28.0 10.3 3.00 0.63 21.1 81.6 1× 16 97 20.3 2.3 2.62 0.06 31.0 98.310× 62 99 6.8 0.3 0.50 0.10 86.8 97.1 100× 95 95 3.3 2.3 0.15 0.06 96.198.3

TABLE 6 R11-AMACH and S-AMACH visual control, height, biomass, andbiomass reduction 3 WAT with various rates of pyrithiobac (POB). VisualControl Height Biomass Biomass Reduction R11-AMACH S-AMACH R11-AMACHS-AMACH R11-AMACH S-AMACH R11-AMACH S-AMACH RATE % cm g % 0 0 0 33.830.5 3.80 3.43 0.0 0.0 1/100× 7 16 26.5 21.8 2.78 1.73 26.8 49.6 1/10×21 59 22.0 8.8 2.42 0.37 36.3 89.2 1× 30 99 17.8 2.0 2.38 0.07 37.4 98.010× 48 99 11.0 1.8 1.07 0.12 71.8 96.5 100× 97 99 2.5 0.5 0.07 0.04 98.298.8

TABLE 7 R11-AMACH and S-AMACH visual control, height, biomass, andbiomass reduction 3 WAT with various rates of cloransulammethyl (TP).Visual Control Height Biomass Biomass Reduction R11-AMACH S-AMACHR11-AMACH S-AMACH R11-AMACH S-AMACH R11-AMACH S-AMACH RATE % cm g % 0 00 46.8 34.4 10.83 8.53 0.0 0.0 1/100× 0 97 47.8 0.7 10.78 0.02 0.5 99.81/10× 23 91 34.5 3.6 8.16 0.25 24.7 97.1 1× 16 96 37.6 1.3 7.49 0.0830.8 99.1 10× 39 99 32.0 0.0 6.44 0.00 40.5 100.0 100× 63 99 12.2 0.02.68 0.00 75.3 100.0

Example 2 Isolation and Sequencing of Herbicide-Resistant ALS Enzymes

To establish why R11-AMACH exhibited high-levels of resistance to fourclasses of ALS-inhibiting herbicides, ALS enzymes from R11-AMACH andS-AMACH were isolated and sequenced. The R11-AMACH nucleotide sequenceis presented in FIG. 1 a (SEQ ID NO: 1) and the corresponding protein inFIG. 1 b (SEQ ID NO: 2). The nucleotide sequence of S-AMACH is presentedin FIG. 2 a (SEQ ID NO: 3) and corresponding protein in FIG. 2 b (SEQ IDNO: 4). No nucleotide differences were observed between R11-AMACH andS-AMACH in any of the five previously reported conserved domains knownto confer ALS resistance in higher plants. However, a single amino aciddifference was discovered in the R11-AMACH biotype ALS that occurred ina conserved region previously unreported to confer ALS resistance inhigher plants (FIG. 3, SEQ ID NO: 5). This region consists of the aminoacid residues, GVRFDDRVTGK, (SEQ ID NO: 6) which are identical to thatof corn (Zea mays), cotton (Gossypium hirsutum), canola (Brassicanapus), rice (Oryza sativa), tobacco (Nicotiana tabacum), andArabidopsis thaliana. The conserved region corresponds to positions 371to 381 of the Arabidopsis ALS coding sequence. At position 375 of thesmooth pigweed ALS amino acid sequence, S-AMACH contained an asparticacid residue, whereas R11-AMACH contained a glutarnic acid residue (FIG.3). The amino acid change was a result of a single point mutation in thenucleotide sequence of R11-AMACH where A replaced T in the sequence GATencoding for aspartic acid (underlined residue is point of mutation).

This invention provides a functional ALS enzyme in higher plants withthe amino acid sequence described in FIG. 1 b, which conferscross-resistance to ALS-inhibiting herbicides comprising four (or more)structurally unrelated chemistries.

Example 3 Enzyme Assay Research

The enzymes from R11-AMACH and S-AMACH were purified and assayed toestablish activity and resistance characteristics on the enzyme level.Purification was accomplished by methodology similar to that of Hill etal., (1997). Briefly, large quantities of the enzyme were produced in anexpression vector in E. coli in which the recombinant protein was fusedto a 6× histidine tag (HIS). Cells were lysed, and the soluble proteinfraction purified by differential centrifugation and subsequentlypassing the protein solution over a nickel column to bind the HIS tag.The ALS protein was eluted from the column, the HIS tag cleaved and thefinal ALS protein purified from small impurities by passage over a sizeexclusion column. Activity was assayed using the discontinuouscolorimetric assay as described by Singh et al. (1988).

The results showed that resistance levels of R11-AMACH enzyme to SU,IMI, POB, TP and sulfonylamino-carbonyl-triazolinone herbicides weregreater than 5 times the concentrations that inhibit the S-AMACH enzyme.

Example 4 Development of Transgenic Crop Plants that are Cross-Resistantto Multiple Herbicides

The mutant ALS gene was amplified from genomic DNA utilizing primersdesigned at the 5′ and 3′ ends to contain both start and stop codons, aswell as restriction sites to be used for ligation into a suitablecloning vector. The complete vector contains an appropriate promoter,antibiotic resistance, and the ALS gene. The complete vector wastransformed into Agrobacterium tumefaciens. The floral dip method(Clough and Bent (1998) was used for Agrobacterium-mediatedtransformation into Arabidopsis thaliana. Seed collected from theseArabidopsis were grown on selective media for transgenic plantselection. Furthermore, R11-AMACH plants germinated and grew normally onmedia containing IMI herbicides at concentrations that completelyinhibited growth of wild-type and S-AMACH plants.

Plants surviving the selective media were grown for seed to evaluateresistance characteristics. ALS-inhibiting herbicides were applied atvarious rates to establish resistance characteristics of the transgenicplants. Transgenic Arabidopsis plants carrying this ALS gene wereresistant to all sulfonylurea, imidazolinone, pyrimidinyloxybenzoate,and triazolopyrimidine herbicides.

Transformation and evaluation of crop plants will follow similar methodsas those employed with Arabidopsis. Soybean and cotton may betransformed, however, with the use of a particle gun to introduceforeign DNA into the genome rather than using Agrobacterium.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

REFERENCES

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1. A substantially purified acetolactate synthase (ALS) enzyme thatconfers, in a plant, cross-resistance to multiple herbicides.
 2. The ALSenzyme of claim 1, wherein the sequence of said ALS enzyme is SEQ ID NO:1, or a fragment thereof with ALS activity.
 3. The ALS enzyme of claim1, wherein at least two of said multiple herbicides are selected fromthe group consisting of sulfonylurea, imidazolinone,pyrimidinyloxybenzoate, triazolopyrimidine andsulfonylamino-carbonyl-triazolinone herbicides.
 4. A substantiallypurified ALS gene encoding an ALS enzyme that confers, in a plant,cross-resistance to multiple herbicides.
 5. The ALS gene of claim 4,wherein said gene is SEQ ID NO: 2 or a fragment thereof encoding apolypeptide with ALS activity.
 6. The ALS gene of claim 4, wherein atleast two of said multiple herbicides are selected from the groupconsisting of sulfonylurea, imidazolinone, pyrimidinyloxybenzoate,triazolopyrimidine and sulfonylamino-carbonyl-triazolinone herbicides.7. A method of conferring cross-resistance to multiple herbicides to aplant, comprising the step of introducing into said plant an expressiblegene encoding an ALS enzyme that exhibits cross-resistance to multipleherbicides, wherein said step of introducing confers cross-resistance tomultiple herbicides to said plant.
 8. The method of claim 7, whereinsaid gene is SEQ ID NO: 1, or a fragment thereof that encodes apolypeptide having ALS activity.
 9. A transgenic plant that iscross-resistant to multiple herbicides, comprised of a host plant thatcontains an expressible gene that is not naturally present in saidplant, said gene encoding an ALS enzyme that confers cross-resistance tomultiple herbicides.
 10. The plant of claim 9, wherein said gene is SEQID NO:2, or a fragment thereof that encodes a polypeptide having ALSactivity.
 11. The transgenic plant of claim 9, wherein at least two ofsaid multiple herbicides are selected from the group consisting ofsulfonylurea, imidazolinoiie, pyrimidinyloxybenzoate, triazolopyrimidineand sulfonylamino-carbonyl-triazolinone herbicides.
 12. The transgenicplant of claim 9 wherein said plant is selected from the groupconsisting of Arabidopsis, corn, cotton, soybean, rice, wheat, andforage crops.
 13. The transgenic plant of claim 9, wherein said ALSenzyme has an aspartic acid to glutamic acid substitution at positionsix of a conserved sequence GVRFDDRVTGK (SEQ ID NO: 6).